Developmental processes involve regulatory cascades among groups of genes that encode DMA binding proteins, which direct the full range of cellular behaviors required for normal function at all life stages. Much remains to be learned about how members of conserved families of DMA binding proteins act in concert to orchestrate patterns of gene expression. This project will investigate one of the most highly conserved of these families, the homeodomain proteins, which are known to be involved in developmental abnormalities and cancer/leukemia progression, among other important disease processes. The broad, long-term goal of these studies is to understand how DMA binding proteins interact with each other and their target sites in the genome to achieve functional specificity during development. The Engrailed homeodomain protein interacts with several other members of the same DNA binding superfamily to help it find and regulate the appropriate target genes. This proposal is to investigate underlying mechanisms using a combination of in vitro DNA binding analysis and the genetic and transgenic methods available in Drosophila, including reporter transgenes, transgenes that generate ectopic protein expression, and rescuing transgenes that mimic the endogenous pattern of expression, each used in both wildtype and mutant genetic backgrounds. The specific aims are: 1) to identify and dissect binding sites for Engrailed within an identified target enhancer in the direct target gene sloppy-paired through a combination of in vitro binding studies and in vivo transgenic analysis;and 2) to dissect the protein domains necessary for Engrailed to cooperate with its cofactors on those target sites, and test the requirements for those domains for in vivo function. PUBLIC HEALTH RELEVANCE: Fundamental knowledge of mechanisms that underlie cellular processes has proven to be essential for developing truly novel approaches to diagnosis and intervention in disease processes of all kinds, most notably in cancer development, where genetic changes accumulating over time are now understood to result in the abnormal cellular behaviors that lead to cancer. These studies will extend our understanding of how genes regulate other genes by studying a family of interacting DNA binding proteins that are highly conserved from Drosophila to humans.